Method for the diagnosis and treatment of cardiovascular diseases

ABSTRACT

The present invention refers to a method for the in vitro or in vivo diagnosis of cardiovascular diseases, in particular high blood pressure, stenosis, vessel occlusion and/or other thrombotic events, wherein the nucleotide at position 950 of a nucleic acid coding for the human ARK2 protein or the amino acid at position 298 of the human ARK2 protein of a sample of a person is determined as well as to the use of ARK2 for the development and/or production of a medicament for treating a cardiovascular disease.

The present invention refers to a method for the in vitro or in vivodiagnosis of cardiovascular diseases, in particular high blood pressure,stenosis, vessel occlusion and/or other thrombotic events, wherein thenucleotide at position 950 of a nucleic acid coding for the human ARK2protein or the amino acid at position 298 of the human ARK2 protein of asample of a person is determined as well as to the use of ARK2 for thedevelopment and/or production of a medicament for treating acardiovascular disease.

The aurora kinases are an oncogenic family of mitotic serine/threoninekinases that are overexpressed in a number of solid tumors(Vankayalapati, H. et al. (2003) Molecular Cancer Therapeutics, 2,283-294). Originally, a spontaneous chromosomal segregation defectmutant of Drosophila was identified and designated aurora (Shindo, M. etal. (1998), 244, 285-292). The human aurora 1 kinase is also known asAUR1, ARK2, Alk2, AIM-1 and STK12. Herein the term ARK2 is used. ARK2shall play a role in mitosis, specifically it accumulates in themidbodies during mitosis (Shindo, M. et al. (1998), supra). ARK2deficient cells have also been shown to exhibit cytokinesis defects(Descamps & Prigent (2001), Sci. STKE, 173, 1). The gene of ARK2 islocated on chromosome 17p13.1.

In order to better understand a potential involvement of ARK2 in theoccurrence and progression of human diseases, genotype-phenotypeassociation analyses have been carried out with a well characterizedpatient group with respect to a C→T variation at position 950 of theARK2 reference sequence published under the reference numberNM_(—)004217. Said variation leads to an amino acid change fromthreonine to methionine (Thr→Met) at the corresponding position 298 inthe ARK2 protein. Different genetic variants of the ARK2 gene arealready known as SNPs (single nucleotide polymorphisms).

Surprisingly it has been found that in particular the variation atposition 950 from cytosine to thymidine in a nucleic acid coding for thehuman ARK2 protein or the corresponding variation of the ARK2 protein atposition 298 from threonine to methionine correlates with the occurrenceof cardiovascular diseases.

Therefore, a subject matter of the present invention relates to an invitro or in vivo diagnosis of cardiovascular diseases, wherein thenucleotide at position 950 of a nucleic acid coding for the human ARK2protein or the amino acid at position 289 of the human ARK2 protein of asample of a person or patient is determined.

In a preferred embodiment of the present invention the cardiovasculardisease is high blood pressure, stenosis, vessel occlusion and/or otherthrombotic events.

In particular, if the nucleotide at position 950 is determined asthymidine in the chromosomal DNA or uracile in the mRNA or the aminoacid at position 298 is determined as methionine there exists a higherrisk of high blood pressure and/or stenosis.

According to the present invention, the term “ARK2-C950C” refers to thegroup of persons which have cytidine on both alleles of the gene codingfor ARK2 at position 950 of the reference sequence NM_(—)004217 whichleads to the amino acid threonine at position 298 of the correspondingprotein. These persons are homozygous with respect to this ARK2 variant.Consequently, the term “ARK2-C950T” refers to the group of persons whohave cytidine on one allele of the gene coding for ARK2 which leads tothreonine at position 298 of the corresponding protein and thymidine onthe other allele of the gene coding for ARK2 which leads to methionineat position 298 of the corresponding protein. These persons areheterozygous with respect to this ARK2 variant. According to the presentinvention, the term “ARK2-T950T” refers to the group of persons whichhave thymidine on both alleles of the gene coding for ARK2 at position950 of the reference sequence NM_(—)004217 which leads to the amino acidmethionine at position 298 of the corresponding protein. These personsare homozygous with respect to this ARK2 variant.

The nucleic acid sequence of the reference sequence coding for the humanARK2 protein preferably has the nucleic acid sequence of SEQ ID NO: 1and the amino acid sequence of the human ARK2 protein preferably has theamino acid sequence of SEQ ID NO: 3. However, the present inventionencompasses also other variants of human ARK2 and the non-human homologsthereof, as for example other mammalian ARK2 homologs or the ARK2homologs from Drosophila, Caenorhabdidis elegans, mouse or rat, providedthat there is a nucleotide exchange from cytidine to thymidine at theposition corresponding to position 950 of said reference sequence and/oran amino acid exchange from threonine to methionine at the positioncorresponding to position 298 of said reference sequence and furtherprovided that the corresponding protein has a serine threonine kinaseactivity. Said enzyme activity can be measured by kinase assays known toa person skilled in the art and/or as described in the presentspecification.

Generally, the specific nucleotide at position 950 can be determined bya nucleic acid sequencing method, a mass spectrometric analysis of thenucleic acid, a hybridisation method and/or an amplification method.Examples of a nucleic acid sequencing method are pyrosequencing and/orsequencing with the help of radioactive and/or fluorescence labellednucleotides. Examples of the hybridisation method are Southern blotanalysis, Northern blot analysis and/or a hybridisation method on aDNA-microarray. Examples of an amplification method are a TaqMananalysis, a differential RNA display analysis and/or a representationaldifference analysis (Shi M. M. (2002) Am J Pharmacogenomics., 2(3),197-205; Kozian & Kirschbaum (1999) Trends Biotechnol., 17(2), 73-8.)

Furthermore, the amino acid sequence at position 298 can be determinedby a method measuring the amount of the specific protein and/or a methodmeasuring the activity of the specific protein. Examples of a method formeasuring the amount of the specific protein are a Western blot analysisand/or an ELISA. Examples for measuring the activity of the specificprotein are an in vitro test assay and/or an in vitro whole cell testassay with human cells, animal cells, bacterial cells or yeast cells,all known to a person skilled in the art and/or described in the presentapplication.

Examples of a sample for the detection of the respective variant are acell, a tissue or a body fluid, in particular in cellular components ofthe blood, endothelial cells or smooth muscle cells. Preferably thesample is pre-treated by conventional methods known to a person skilledin the art in order to isolate and/or purify the nucleic acids orchromosomal DNA, or the proteins of the sample for the further analysis.

In an optional further step the risk of a person to suffer from acardiovascular disease can be determined as shown in the examples.

In another optional further step an appropriate pharmaceutical isselected or the dosage of a pharmaceutical is determined.

In general, the found genetic variation in the ARK2 gene can be used inaccordance with the present invention as a genetic marker for the riskassessment, the genetic characterization or classification of a personand/or the prophylactic treatment of a cardiovascular disease (alsoknown as “coronary heart disease”), in particular of high bloodpressure, stenosis, vessel occlusion and/or other thrombotic events.

Furthermore, the genetic variation can be used in accordance with thepresent invention as a genetic marker for the adaptation of the dosageor generally of increasing the effectiveness of an effective therapeuticagent for the treatment of a person or patient (hereinafter alsoreferred to as “individual”) and/or for the identification ofindividuals being under or selected to be under clinical trial studieswith an increased risk for cardiovascular disease, in particular of highblood pressure and/or stenosis. The genetic variation can also be usedin accordance with the present invention for the evaluation of thetolerance, safety and efficacy of a pharmaceutically active substancefor a specific individual or for identifying the individual suitable fora particular treatment of said diseases.

The present invention can also be used to identify risk factors for saiddiseases for each individual to be treated or advised.

In general, suitable individuals are (i) individuals who do not exhibitthe symptoms of a cardiovascular disease, (ii) individuals in whom therisk of developing a cardiovascular disease has already been detectedbut who do not yet exhibit the symptoms of the disease, and (iii)individuals who have previously been diagnosed as suffering from acardiovascular disease.

A preferred method for the diagnosis of a cardiovascular disease inaccordance with the present invention contains the following steps:

-   -   (a) optionally obtaining a sample, in particular a cell, tissue,        body fluid, a cellular component of the blood, endothelial cells        or smooth muscle cells, from a person or patient that should be        investigated;    -   (b) isolating a nucleic acid probe, in particular a DNA probe        from the sample;    -   (c) amplifying the specific region encompassing position 950 of        the ARK2 gene with the help of primers, in particular the        primers as specified in the Examples;    -   (d) sequencing the amplified region;    -   (e) analysing the sequenced region; and    -   (f) assessing the risk for a cardiovascular disease, in        particular for high blood pressure, stenosis, vessel occlusion        and/or other thrombotic events.

An alternative method for the diagnosis of a cardiovascular disease inaccordance with the present invention contains the following steps:

-   -   (a) optionally obtaining a sample, in particular a cell, tissue,        body fluid, a cellular component of the blood, endothelial cells        or smooth muscle cells, from a person or patient that should be        investigated;    -   (b) isolating the ARK2 protein from the sample;    -   (c) determining the amino acid at position 298 of the ARK2        protein; and    -   (d) assessing the risk for a cardiovascular disease, in        particular for high blood pressure, stenosis, vessel occlusion        and/or other thrombotic events.

The present invention generally refers also to a method for determiningthe risk of a person to suffer from a cardiovascular disease, whereinthe method comprises the steps of

-   -   (a) determining the genotype of the ARK2 gene of a person in        vitro or in vivo, and    -   (b) converting the data obtained in (a) in order to give a        prognosis for said person's risk of developing a cardiovascular        disease,        whereas the detection of an ARK2 Met298Met variation is an        indicator for an increased risk for developing a cardiovascular        disease, in particular high blood pressure, stenosis, vessel        occlusion and/or other thrombotic events.

Another embodiment of the present invention generally refers to a methodfor selecting a pharmaceutically active compound or determining thedosage of a pharmaceutically active compound for a person suffering froma cardiovascular disease, in particular high blood pressure, stenosis,vessel occlusion and/or other thrombotic events, or for determining theeffectiveness of a therapeutic treatment of a cardiovascular disease, inparticular high blood pressure, stenosis, vessel occlusion and/or otherthrombotic events, wherein the method comprises the steps of

-   -   (a) determining the genotype of the ARK2 gene of a person in        vitro, and    -   (b) selecting a pharmaceutically active compound or determining        the dosage of a pharmaceutically active compound for said person        or determining the effectiveness of a therapeutic treatment,        whereas the pharmaceutically active compound is selected and/or        the dosage of a pharmaceutically active compound and/or the        effectiveness of a therapeutic treatment is determined for a        person having an ARK2 Met298Met variation.

Still another embodiment of the present invention generally refers to amethod for identifying a person having an increased risk for acardiovascular disease, wherein the method comprises the steps of

-   -   (a) determining the genotype of the ARK2 gene of a person in        vitro or in vivo, and    -   (b) converting the data obtained in (a) in order to identify the        person,        whereas the detection of an ARK2 Met298Met variation is an        indicator for an increased risk for developing a cardiovascular        disease, in particular high blood pressure, stenosis, vessel        occlusion and/or other thrombotic events.

The determination of the genotype of the ARK2 gene can be carried out byany method known to a person skilled in the art, in particular by anymethod described herein.

In addition, ARK2 and/or the ARK2 variants as described herein can beused in accordance with the present invention for the production of amedicament for treating a cardiovascular disease. Therefore, anadditional embodiment of the present invention refers to the use of anARK2 protein containing an amino acid sequence according to SEQ ID NO: 3or of an ARK2 variant at the amino acid position 298 and/or thecorresponding nucleic acid sequence coding for the ARK2 protein orvariant thereof for the production of a medicament for treating acardiovascular disease, in particular high blood pressure, stenosis,vessel occlusion and/or other thrombotic events. In particular, thevariant is a Met298Met variant of the ARK2 protein according to SEQ IDNO: 7 or a T950T variant of the ARK2 nucleic acid according to SEQ IDNO: 6. In particular, kinase assays known to those of skill in the artand/or as described herein can be used to identify modulators, e.g.activators or inhibitors, of the ARK2 protein and/or the ARK2 variant,in particular the ARK2-Met298Met variant.

Consequently, ARK2 and/or the ARK2 variants described herein can also beused in accordance with the present invention as part of a highthroughput-screening assay for the detection and evaluation ofpharmaceutically active compounds for the treatment of said diseases.Therefore, an additional embodiment of the present invention refers to amethod of screening a pharmaceutically active agent for the treatment ofa cardiovascular disease, wherein the method comprises the steps of:

-   -   (a) providing an ARK2 protein containing an amino acid sequence        according to SEQ ID NO: 3 or an ARK2 variant at the amino acid        position 298 and/or the corresponding nucleic acid coding for        the ARK2 protein or variant thereof,    -   (b) providing a test compound,    -   (c) measuring or detecting the influence of the test compound on        the ARK2 protein or ARK2 variant or on the corresponding nucleic        acid, and    -   (d) isolating a compound suitable for the treatment of a        cardiovascular disease, in particular high blood pressure,        stenosis, vessel occlusion and/or other thrombotic events.

In particular, the variant is a Met298Met variant of the ARK2 proteinaccording to SEQ ID NO: 7 or a T950T variant of the ARK2 nucleic acidaccording to SEQ ID NO: 6. In general, the ARK2 protein, the ARK2variant or the nucleic acid coding for the ARK2 protein or variant isprovided e.g. in an assay system and brought directly or indirectly intocontact with a test compound, in particular a biochemical or chemicaltest compound, e.g. in the form of a chemical compound library. Then,the influence of the test compound on the ARK2 protein or the nucleicacid coding for the ARK2 protein is measured or detected. Thereafter,suitable modulators, e.g. activators or inhibitors, can be analyzedand/or isolated. For the screening of chemical compound libraries, theuse of high-throughput assays are preferred which are known to theskilled person or which are commercially available.

In general, the influence of the test compound on the ARK2 protein orthe ARK2 variant or the nucleic acid coding for the ARK2 protein or thevariant may be any physical, chemical or phenotypic effect of thecompound upon the protein or nucleic acid or upon a cell comprising theprotein or nucleic acid, thereby identifying a compound that modulatesthe protein or nucleic acid. In the present case it is preferable tomeasure or detect the influence of the test compound on the kinaseactivity of the ARK2 protein or the ARK2 variant as described herein.

The general concept of a kinase assay is that the kinase to be analysed,here the ARK2 serine/threonine kinase, is brought into contact with asuitable substrate or peptide containing a serine or threonine residuewhich can be phosphorylated by the kinase in the presence of preferablyATP in a suitable buffer. Preferably the substrate is a dye-labelledsubstrate, e.g. a fluorescent dye-labelled peptide, e.g. afluorescein-labelled peptide. Serine/Threonine kinase assays arecommercially available, e.g. the HitHunter™ Serine/Threonine KinaseAssay from Applied Biosystems, Inc., California, USA or the IQ™Serine/Threonine Kinase Assay from Pierce Biotechnology, Inc., Illinois,USA. Other kinase assays are further described in detail below.

According to the present invention the term “chemical compound library”refers to a plurality of chemical compounds that have been assembledfrom any of multiple sources, including chemically synthesized moleculesand natural products, or that have been generated by combinatorialchemistry techniques.

In general, the influence of the test compound on ARK2, the ARK2 variantor the nucleic acid coding for ARK2 protein or variant is measured ordetected in a heterogeneous or homogeneous assay. As used herein, aheterogeneous assay is an assay which includes one or more washingsteps, whereas in a homogeneous assay such washing steps are notnecessary. The reagents and compounds are only mixed and measured.

Suitable functional assays may be based on the gene expression of ARK2or its serine/threonine kinase activity. In general, commerciallyavailable kinase assays systems quantitatively detect the amount ofphosphate incorporated in a substrate.

Heterogeneous assays are, for example, ELISA, DELFIA, SPA and flashplateassays.

ELISA (enzyme linked immuno sorbent assay)-based assays are offered byvarious companies. The assays employ random peptides that can bephosphorylated by a kinase, such as ARK2. Kinase-containing samples areusually diluted into a reaction buffer containing e.g. ATP and requisitecations and then added to plate wells. Reactions are stopped by simplyremoving the mixtures. Thereafter, the plates are washed. The reactionis initiated e.g. by the addition of a biotinylated substrate to thekinase. After the reaction, a specific antibody is added. The samplesare usually transferred to pre-blocked protein-G plates and afterwashing e. g streptavidin-HRP is added. Thereafter, unboundstreptavidin-HRP (horseradish peroxidase) is removed, the peroxidasecolour reaction is initiated by addition of the peroxidase substrate andthe optical density is measured in a suitable densitometer.

DELFIA (dissociation enhanced lanthanide fluoro immuno assay)-basedassays are solid phase assay. The antibody is usually labelled withEuropium or another lanthanide and the Europium fluorescence is detectedafter having washed away un-bound Europium-labelled antibodies.

SPA (scintillation proximity assay) and the flashplate assay usuallyexploit biotin/avidin interactions for capturing radiolabelledsubstrates. Generally the reaction mixture includes the kinase, abiotinylated peptide substrate and γ-[P³³]ATP. After the reaction, thebiotinylated peptides are captured by streptavidin. In the SPAdetection, streptavidin is bound on scintillant containing beads whereasin the flashplate detection, streptavidin is bound to the interior ofthe well of scintillant containing microplates. Once immobilized, theradiolabelled substrate is close enough to the scintillant to stimulatethe emission of light.

Alternative homogeneous assays are, for example, TR-FRET, FP, ALPHA andgene assays.

TR-FRET (time-resolved fluorescence resonance energy transfer)-basedassays are assays which usually exploit the fluorescence resonanceenergy transfer between Europium and APC, a modified allophycocyanin orother dyes with overlapping spectra such as Cy3/Cy5 or Cy5/Cy7 (Schobel,U. et al. (1999) Bioconjugate Chem. 10, 1107-1114). After excitatione.g. of Europium with light at 337 nm, the molecule fluoresces at 620nm. But if this fluorophore is close enough to APC, the Europium willtransfer its excitation energy to APC, which fluoresces at 665 nm. Thekinase substrate is usually a biotin-labelled substrate. After thekinase reaction, Europium-labelled-(P)-specific antibodies are addedalong with streptavidin-APC. The phosphorylated peptides bring theEuropium-labelled antibody and the streptavidin-APC into close contact.The close proximity of the APC to the Europium fluorophore will cause aquenching of the Europium fluorescence at benefit of the APCfluorescence (FRET).

Fluorescence polarisation (FP)-based assays are assays which usepolarized light to excite fluorescent substrate peptides in solution.These fluorescent peptides are free in solution and tumble, causing theemitted light to become depolarised. When the substrate peptide binds toa larger molecule, however, such as (P)-Tyr, its tumbling rates aregreatly decreased, and the emitted light remains highly polarized. For akinase assay there are generally two options:

-   -   (a) A fluorescent phosphopeptide tracer is bound to a        (P)-specific antibody. Phosphorylated products will compete the        fluorescent phosphopeptide from the antibody resulting in a        change of the polarisation from high to low.    -   (b) A phosphorylated substrate peptide binds to the        phosphospecific antibody resulting in a change of polarisation        from low to high.        ALPHA (amplified luminescent proximity homogenous)-based assays,        are assays which rely on the transfer of singlet oxygen between        donor and acceptor beads brought into proximity by a        phosphorylated peptide. Upon excitation at 680 nm,        photosensitisers in donor beads convert ambient oxygen to        singlet-state oxygen, which diffuses up to a distance of 200 nm.        Chemiluminescent groups in the acceptor beads transfer energy to        fluorescent acceptors within the bead, which then emits light at        approximately 600 nm.

EFC (enzyme fragment complementation)-based assays or equivalent assayscan be used in particular for high-throughput screening of compounds.The EFC assay is based on an engineered β-galactosidase enzyme thatconsists of two fragments—the enzyme acceptor (EA) and the enzyme donor(ED). When the fragments are separated, there is no β-galactosidaseactivity, but when the fragments are together they associate(complement) to form active enzyme. The EFC assay utilizes an ED-analyteconjugate in which the analyte may be recognized by a specific bindingprotein, such as an antibody or receptor. In the absence of the specificbinding protein, the ED-analyte conjugate is capable of complementing EAto form active β-galactosidase, producing a positive luminescent signal.If the ED-analyte conjugate is bound by a specific binding protein,complementation with EA is prevented, and there is no signal. If freeanalyte is provided (in a sample), it will compete with the ED-analyteconjugate for binding to the specific binding protein. Free analyte willrelease ED-analyte conjugate for complementation with EA, producing asignal dependent upon the amount of free analyte present in the sample.

Another example of a gene assay is a functional assay wherein theactivity of the kinase is converted into a functional cellular responsesuch as growth, growth arrest, differentiation or apoptosis. For thistype of screening yeast is a particularly suitable model system. Forexample in an ARK2-yeast functional assay, when cultured on glucosecontaining medium, the e.g. ARK2-yeast cells grow like normal yeastcells. When, however being exposed to galactose, the intracellularexpression of ARK2 is induced causing the yeast cell to die. Compoundsthat inhibit ARK2 activity prevent the cell death in this case.

Another assay is based on solid phase-bound polypeptides such as ARK2and the interference with the compounds to be tested. Thus, a testcompound contains a detectable marker, for example, the compound can beradioactively labelled, fluorescence-labelled or luminescence-labelledas already explained above. Furthermore, compounds can be coupled toproteins which permit indirect detection, for example by means ofenzymatic catalysis employing a peroxidase assay which uses achromogenic substrate or by means of binding a detectable antibody.Another possibility is that of investigating the solid phase-boundprotein complexes by means of mass spectrometry (SELDI). Changes in theconformation of e.g. ARK2 or the ARK2 variants described herein as theresult of interaction with a test substance can be detected, forexample, by the change in the fluorescence of an endogenous tryptophanresidue in the polypeptide.

The solid phase-bound polypeptides can also be part of an array. Methodsfor preparing such arrays using solid phase chemistry and photolabileprotecting groups are disclosed, for example, in U.S. Pat. No.5,744,305. These arrays can also be brought into contact with testcompound or compound libraries and tested for interaction, for examplebinding or changing conformation. Suitable formats of the arrays arecurrently in the 96-, 384- or 1,536 formats for both the primary andsecondary screens.

In another embodiment of the present invention, the method is carriedout using whole cells. Usually cells growing at the bottom of multiwellplates are fixed and permeabilized, blocked and incubated with e.g. aprimary (P)-specific antibody against the substrate of interest. Then,e.g. Europium labelled or HRP conjugated secondary antibodies inconjunction with specific chemiluminescent or colorimetric substances,e.g. as described above, are utilized to generate the signal. Incombination with the use of a microscope not only the amount of(P)-specific antibodies can be quantified on the single cell level, butalso phosphorylation-induced translocations of a substrate ormorphological changes of the cells.

Advantageously the method of the present invention is carried out in arobotics system e.g. including robotic plating and a robotic liquidtransfer system, e.g. using microfluidics, i.e. channelled structured.

In another embodiment of the present invention, the method is carriedout in form of a high-through put screening system. In such a systemadvantageously the screening method is automated and miniaturized, inparticular it uses miniaturized wells and microfluidics controlled by aroboter.

In view of the above subject matter the present invention refers also toa method for producing a medicament for the treatment of acardiovascular disease, in particular high blood pressure, stenosis,vessel occlusion and/or other thrombotic events, wherein the methodcomprises the steps of:

-   -   (a) carrying out the screening method as explained above, and    -   (b) formulating the isolated compound with one or more        pharmaceutically acceptable carriers or auxiliary substances.

According to step (b) of the above method the detected test compound isusually formulated with one or more pharmaceutically acceptableadditives or auxiliary substances, such as physiological buffersolution, e.g. sodium chloride solution, demineralized water,stabilizers, ε-aminocaproic acid or pepstatin A or sequestering agentssuch as EDTA, gel formulations, such as white vaseline, low-viscosityparaffin and/or yellow wax, etc. depending on the kind ofadministration.

Suitable further additives are, for example, detergents, such as, forexample, Triton X-100 or sodium deoxycholate, but also polyols, such as,for example, polyethylene glycol or glycerol, sugars, such as, forexample, sucrose or glucose, zwitterionic compounds, such as, forexample, amino acids such as glycine or in particular taurine or betaineand/or a protein, such as, for example, bovine or human serum albumin.Detergents, polyols and/or zwitterionic compounds are preferred.

The physiological buffer solution preferably has a pH of approx.6.0-8.0, especially a pH of approx. 6.8-7.8, in particular a pH ofapprox. 7.4, and/or an osmolarity of approx. 200-400 milliosmol/liter,preferably of approx. 290-310 milliosmol/liter. The pH of the medicamentis in general adjusted using a suitable organic or inorganic buffer,such as, for example, preferably using a phosphate buffer, tris buffer(tris(hydroxymethyl)aminomethane), HEPES buffer([4-(2-hydroxyethyl)piperazino]-ethanesulphonic acid) or MOPS buffer(3-morpholino-1-propanesulphonic acid). The choice of the respectivebuffer in general depends on the desired buffer molarity. Phosphatebuffer is suitable, for example, for injection and infusion solutions.

The medicament can be administered in a conventional manner, e.g. bymeans of oral dosage forms, such as, for example, tablets or capsules,by means of the mucous membranes, for example the nose or the oralcavity, in the form of dispositories implanted under the skin, by meansof injections, infusions or gels which contain the medicaments accordingto the invention. Furthermore, the treatment can be carried out by meansof a transdermal therapeutic system (TTS), which makes possible atemporally controlled release of the medicaments. TTS are known forexample, from EP 0 944 398 A1, EP 0 916 336 A1, EP 0 889 723 A1 or EP 0852 493 A1.

Injection solutions are in general used if only relatively small amountsof a solution or suspension, for example about 1 to about 20 ml, are tobe administered to the body.

Infusion solutions are in general used if a larger amount of a solutionor suspension, for example one or more litres, are to be administered.Since, in contrast to the infusion solution, only a few millilitres areadministered in the case of injection solutions, small differences fromthe pH and from the osmotic pressure of the blood or the tissue fluid inthe injection do not make themselves noticeable or only make themselvesnoticeable to an insignificant extent with respect to pain sensation.Dilution of the formulation according to the invention before use istherefore in general not necessary. In the case of the administration ofrelatively large amounts, however, the formulation according to theinvention should be diluted briefly before administration to such anextent that an at least approximately isotonic solution is obtained. Anexample of an isotonic solution is a 0.9% strength sodium chloridesolution. In the case of infusion, the dilution can be carried out, forexample, using sterile water while the administration can be carriedout, for example, via a so-called bypass.

More preferred steps are individually or collectively specified in theExamples and are incorporated hereby by reference to each step.

The following Figures, Tables, Sequences and Examples shall explain thepresent invention without limiting the scope of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1A shows the nucleic acid sequence of the human ARK2 gene with theNCBI number NM_(—)004217.

FIG. 1B shows part of the chromosomal nucleic acid sequence of ARK2 withthe number AC135178. The primers used for amplification of the geneticsection with the genetic variation C→T corresponding to position 950 ofreference sequence NM_(—)004217 are marked in bold face and underlined(ARK2-298R=CCTGCAGGTGGACCTAAAGTTC; ARK2-298F=GCCTGAGCAGTTTGGAGATGAG).

FIG. 2 shows the amino acid sequence of the human ARK2 derived from thenucleic acid sequence with the NCBI number NM_(—)004217. The amino acidposition 298 in the ARK2 protein is in bold face and underlined.

FIG. 3 shows the nucleic acid sequence of the human ARK2 gene T950Tvariant.

FIG. 4 shows the amino acid sequence of the human ARK2 Met298Metvariant.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 shows the nucleic acid sequence of the human ARK2 proteinwith the NCBI number NM_(—)004217.

SEQ ID NO: 2 shows part of the chromosomal nucleic acid sequence of ARK2with the number AC135178.

SEQ ID NO: 3 shows the amino acid sequence of the human ARK2 derivedfrom the nucleic acid sequence with the NCBI number NM_(—)004217.

SEQ ID NO: 4 shows the first primer sequence ARK2-298R.

SEQ ID NO: 5 shows the second primer sequence ARK2-298F of thecomplementary sequence.

SEQ ID NO: 6 shows the nucleic acid sequence of the human ARK2 geneT950T variant.

SEQ ID NO: 7 shows the amino acid sequence of the human ARK2 Met298Metvariant.

EXAMPLES

SNP Detection by 5′-Nuclease Assays (TaqMan™)

Oligonucleotides (Primers) for Amplification:

The following primers were used for the detection of the nucleotideexchange from C to T at position 950 in the ARK2 sequence with thereference number NM_(—)004217:

Primer 1: 5′-GCCTGAGCAGTTTGGAGATGAG-3′ (nucleotides 179605-179584 of thereference sequence AC135178; FIG. 1B; SEQ ID NO: 5);

Primer 2: 5′-CCTGCAGGTGGACCTAAAGTTC-3′ (complementary sequence of bases179532-179553 of the reference sequence AC135178; FIG. 1B; SEQ ID NO:4).

PCR Protocol for Amplification:

The reagents used were from Applied Biosystems (Foster City, USA). ThePCR reaction was carried out in a Primus 96 plus thermal cycler (MWGBiotech AG, Germany) in a total volume of 5 μl with 2.5 μlSuperHot-Master-Mix (Bioron GmbH, Germany), 0.125 μl Assay-by-Design-Mix(Applied Biosystems, Austria), 0.375 μl H₂O and 2 μl DNA. The reactionmixtures were overlaid by 15 μl mineral oil.

Amplification Program of the PCR reactions:

-   94° C. for 1 min×1 cycle-   92° C. for 15 sec×45 cycles-   60° C. for 1 min×1 cycle;

Analysis of the PCR Products

The fluorescence was detected in a VICTOR Fluorescence Plate Reader (HVDLife Sciences, Austria) with an excitation/emission filter of 485 nm/520nm for FAM-marked probes (298Thr allele; ARK2-298M1 (Thr)FAM-TCCCGTGGGCACG) and 530 nm/572 nm for VIC-marked probes (298Metallele; ARK2-298V1 (Met) VIC-CTCCCATGGGCACG). The data were exported ina MS-Excel format and analysed with scatter plots.

Results

Characteristics of the Group of Persons

Table 1 shows the characteristics of the group of persons studied.

TABLE 1 n % Total 2074 Sex Female 603 29.07 Male 1471 70.93 Age  61.8(+/− 10.5) BMI (Body Mass 29.1 (+/− 4.4) Index) Blood Pressure 1214 58.7Smoker 1372 66.41 Type II Diabetes 361 17.46 Myocardial infarction 83040.59 Stroke 145 7.01

Frequence and Distribution of the Variants of the ARK2 Gene

Table 2 shows the frequency and distribution of the genetic variants ofthe ARK2 gene at position 950 of the reference sequence NM_(—)004217 inthe patient group studied.

TABLE 2 Frequency Percentage ARK2-T950T 21 1.46 (ARK2 Met298Met)ARK2-C950T 290 20.18 (ARK2 Thr298Met) ARK2-C950C 1126 78.36 ARK2Thr298Thr)

Influence of the Variant Thr298Met of ARK2

Table 3 shows the influence of the variant Thr298Met of ARK2 on theoccurrence of high blood pressure, coronary heart diseases (with astenosis of >20%), the incidence of more than one myocardial infarctionand stroke/TIA/PRIND (TIA=transitoric ischemic attack; PRIND=prolongedreversible ischemic neurological deficit) in the patient group studied.P-values less than 0.05 are considered to be statistically relevant.

TABLE 3 ARK2- ARK2- ARK2- T950T C950T C950C p P value Met298MetThr298Met Thr298Thr value adjusted High blood pressure 16 (76.19%) 184(63.45%) 618 (54.88%) 0.0063 Coronary heart 17 (85.00%) 238 (82.93%) 859(77.81%) 0.0286 diseases >1 Myocardial  3 (14.29%) 28 (9.66%) 66 (5.86%)0.0464 infarction Stroke/TIA/PRIND  6 (28.57%) 17 (5.86%) 83 (7.37%)0.0006

Results:

The patients with ARK2-T950T showed a statistically higher incidence forhigh blood pressure and coronary heart diseases, the incidence of morethan one myocardial infarction and stroke/TIA/PRIND compared to patientswith different ARK2 genotype at this position 950.

Conclusion:

The statistically significant associations between the genetic variantsof the gene coding for ARK2 and/or the protein ARK2 shown above are aclear indication for the involvement of said genetic variants in theoccurrence of cardiovascular diseases, in particular high bloodpressure, stenosis, vessel occlusion and/or thrombotic events.Consequently, said genetic variants are biological markers for e.g. theprognosis of cardiovascular diseases, in particular high blood pressure,stenosis, vessel occlusion and/or thrombotic events). Furtherembodiments of the invention based on the present finding are describedin further details in the present specification.

1. A method for determining the risk of occurrence of a cardiovasculardisease in an individual comprising the steps of: (a) obtaining a samplefrom said individual, (b) isolating nucleic acid from said sample, (c)amplifying the specific region in said nucleic acid encompassingposition 950 of the ARK2 gene with the help of primers; (d) sequencingthe amplified region; (e) analysing the sequenced region wherein thedetermination of thymidine or uracile at position 950 is indicative ofincreased risk for a cardiovascular disease.
 2. The method of claim 1wherein the cardiovascular disease comprises the group consisting ofhigh blood pressure, stenosis, vessel occlusion and thrombotic events.3. The method according to claim 1 wherein the nucleic acid coding forthe human ARK2 protein has the nucleotide sequence of SEQ ID NO:
 6. 4.The method according to claim 1 wherein the nucleotide at position 950is determined by a method selected from the group consisting of anucleic acid sequencing method, a mass spectrometric analysis of thenucleic acid, a hybridization method and an amplification method.
 5. Themethod according to claim 1, wherein said nucleic acid sequencing methodis selected from the group selected of pyrosequencing and sequencingusing radioactive and fluorescence labeled nucleotides.
 6. The methodaccording to claim 4 wherein said hybridization method is selected fromthe group consisting of Southern blot analysis, Northern blot analysisand a hybridization method on a DNA-microarray.
 7. The method accordingto claim 4 wherein said amplification method is selected from the groupconsisting of a TaqMan analysis, a differential RNA display analysis anda representational difference analysis.
 8. A method for determining therisk of occurrence of a cardiovascular disease in an individualcomprising the steps of comprising the steps of (a) obtaining a samplefrom a person, (b) isolating the ARK2 protein from the sample, (c)determining the amino acid at position 298 of the ARK2 protein, and (d)assessing the risk for a cardiovascular disease wherein the detection ofan ARK2 Met298Met variation is an indicator for an increased risk fordeveloping a cardiovascular disease.
 9. The method according to claim 8wherein said sample comprises the group consisting of a cell, tissue,body fluid, a cellular component of the blood, endothelial cells andsmooth muscle cells.
 10. The method according to claim 8 wherein saidcardiovascular disease is selected from the group consisting of highblood pressure, stenosis, vessel occlusion and thrombotic events. 11.The method according to claim 8 wherein the amino acid sequence atposition 298 is determined by a method selected from the groupconsisting of a method measuring the amount of the specific protein anda method measuring the activity of the specific protein.
 12. The methodof claim 11 wherein said activity is selected from group consisting ofserine and threonine kinase activity.
 13. The method according to claim11 wherein said amount of the specific protein is measured by a methodselected from the group consisting of a western blot analysis and anELISA.
 14. The method according to claim 11 wherein said activity of thespecific protein is measured by the group consisting of an in vitro testassay and an in vitro whole cell test assay with human cells, animalcells, bacterial cells or yeast cells.